Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
repeated dose toxicity: oral
Remarks:
other: reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 December 2011 - 26 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.
Deviations:
yes
Remarks:
Additional liver and heamatology parameters were evaluated.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
- Name of test material (as cited in study report): Salicynalva
- Substance type: Clear colourless to pale yellow liquid
- Physical state: liquid
- Storage condition of test material: At room temperature in the dark
- Volatile: Not indicated
- Relative density: 0.9460
- Stability at higher temperatures: Not indicated
- Vapour pressure: 6.4 Pa

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 12 weeks.
- Weight at study initiation: mean weight at start of treatment was 321 - 325 gr (males) or 203 - 204 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle.

Temporary deviations from the minimum level of relative humidity occurred.
Evaluation: Laboratory historical data do not indicate an effect of the deviations.

IN-LIFE DATES: From: 27 December 2011 to 26 April 2012

Administration / exposure

Route of administration:
oral: feed
Details on oral exposure:
- Method of formulation: Oral, by inclusion in the diet.
- Dietary Inclusion Levels: The amount of test substance incorporated into the diet was kept at a constant level in terms of ppm, throughout the study period. After termination, the actual test substance intake was estimated based on the body weight and food consumption values.
- Storage conditions of diets: In the freezer until day of use. Twice weekly the pellets were defrosted and offered to the animals for a maximum of four days.
- Stability of pelleted diet: At least 14 days stable in the freezer and 4 days at room temperature (concentration range of 200 to 4.500 ppm; determined during NOTOX Project 498293).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (07 March 2012), according to a validated method (NOTOX Project 498293). Dietary samples were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).

In addition, random samples were taken and stored at ≤-15°C for possible future analysis. Any remaining samples will be discarded after approval by the sponsor, or at finalization of the study report.

The accuracy of diet preparations was considered acceptable if the mean measured concentrations were 80-120% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

The concentrations analysed in the diets of Group 2, Group 3 and Group 4 were below target concentrations (i.e. mean accuracies between 67% and 77%). This was probably due to loss of test substance during preparation of the pellets (volatilized).

A small response at the retention time of the test substance was observed in the chromatograms of the Group 1 diet. The maximum contribution to the samples based on area was 0.6%.

The diets of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

For the diets of Groups 2, 3 and 4 a mean accuracy of 67%, 71% and 77%, respectively, was obtained, which was below the criterion range 80-120%.
Evaluation: As no treatment related findings were noted up to the highest dose level tested (1.000 ppm) and as this level was just below the criterion range (77% instead of 80%), it was decided this slight deviation was negligible.
Duration of treatment / exposure:
Males were exposed for 32 days, i.e. 2 weeks prior to mating, during mating until scheduled necropsy. Females were exposed for 43-56 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 5 days of lactation.
Frequency of treatment:
Ad libitum.
Doses / concentrations
Remarks:
Doses / Concentrations:
15, 30 and 70 mg/kg bw (nominal doses were 0, 200, 400, 1000 ppm i n the diet)
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose levels were based on results of a 14-Day dose range finding study (See attached results).
Based on the results of this dose range finding study, dose levels for the main study were selected by the sponsor and determined to be 200, 400 and 1.000 ppm.
Positive control:
No.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Daily, detailed clinical observations were made in all animals. The time of onset, grade and duration of any observed sign was recorded.

BODY WEIGHT:
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

FOOD CONSUMPTION:
- Twice weekly, except for males and females which were housed together for mating and for females without evidence of mating.
Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY:
- (average food consumption [per animal per day]/average body weight per cage)x1000

WATER CONSUMPTION : No.
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood: between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: No.
- How many animals: 10 animals/sex/group
- Parameters checked were: According to test guidelines

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

On Day 1 of lactation, an unrealistic high value for body weight was collected for one female from the control group; this value was excluded from the tables.
Evaluation: Sufficient information available for evaluation.

Clinical signs observations were not performed for one female from the control group on Day 3 of lactation.
Evaluation: Sufficient information available for evaluation.

Observations of pups were not recorded online on the following occasions:
Day 4 of lactation for one litter from the control group, two litters from Group 3, and on Day 5 of lactation for one pup of one litter from Group 2 and one pup of one litter from Group 3.
Evaluation: Body weight of these pups was determined on Day 4 and therefore these pups were also observed at that moment. In addition, no symptoms were recorded the day before and the day following this omission. Sufficient data is available for a thorough evaluation.

One pup of one litter from the control group was not observed and weighed on Day 1 of lactation. It was not recorded if one pup of one litter from Group 2 went into necropsy. In addition, for 11 pups of one litter from the control group external examination at necropsy was not recorded.
Evaluation: No findings were noted on the other days. Sufficient information available for evaluation.

Sacrifice and pathology:
GROSS PATHOLOGY:
- The animals were not deprived of food. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- According to test guidelines

ORGAN WEIGHTS
-Epididymides, Kidneys, Liver, Spleen and Testes

HISTOPATHOLOGY:
- According to test guidelines
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher, 1950) was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
As no microscopic correlate was noted for the increased liver weights at 1.000 ppm, it was not considered toxicologically significant.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
MORTALITY
No mortality occurred during the study period.

CLINICAL SIGNS
No treatment related clinical signs were noted up to 1.000 ppm.

One female at 200 ppm showed swelling of the right foreleg for several days and one female at 400 ppm showed piloerection for one day. At this low incidence and as it was not noted at 1.000 ppm, it was not considered toxicologically significant.

BODY WEIGHTS
For all dose levels, body weights and body weight gain remained in the same range as controls over the treatment period.

One female at 400 ppm showed a body weight loss of 9% over Days 1 to 4 of lactation. At this single occurrence at the mid dose level, it was not considered toxicologically relevant.

FOOD CONSUMPTION
No toxicologically relevant findings were noted for food consumption before or after allowance for body weight for animals treated up to 1.000 ppm.

The statistical significant changes noted for treated females (all dose levels) during post-coitum and/or lactation were not considered toxicologically relevant as all values were well within normal limits of which the concurrent control group at the lower end.

The female at 1000 ppm that showed a severe body weight loss during lactation also showed a severely reduced food consumption during that period. This was not considered toxicologically relevant, as it was only noted for one animal at the mid dose.

HAEMATOLOGY
For haematology parameters, no treatment related findings were noted up to 1.000 ppm for both sexes.

Two females of the control group showed a high percentage of neutrophils and eosinophils with concurrent low percentage of lymphocytes. All other parameters determined for these females were within normal limits, and it was therefore not considered toxicologically significant. Moreover, as it concerned two control animals it was not treatment related.

MACROSCOPIC EXAMINATION
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.

Incidental findings consisted of pelvic dilation of the kidneys, nodule at the epididymides, discolouration of the preputial or clitoral glands, enlarged liver or spleen, fluid in the uterus, and spleen reduced in size. These findings were within the background range of findings that are encountered among rats of this age and strain and did not show a dose-related incidence trend, and were therefore not considered to be of any toxicological relevance.

ORGAN WEIGHTS
Increased liver weights were noted at 1.000 ppm for both sexes. This was statistically significant for absolute liver weight for the females and for absolute and relative liver weights for the males.

No treatment related effects were noted for the weights of the kidneys, spleen, testes and epididymides up to 1.000 ppm.

MICROSCOPIC EXAMINATION
There were no treatment related microscopic observations. Recorded findings were considered to be within the normal range of background pathology encountered in Wistar (Han) rats of this age and strain. There was no correlate to the statistically significant increased group mean liver weights of 1.000 ppm animals.

No abnormalities were seen in the reproductive organs of the pair of Group 3 animals that failed to conceive. There was evidence that the female was cycling. Staging of spermatogenesis did not provide any evidence of test article related impairment to the spermatogenetic cycle.

Effect levels

Dose descriptor:
NOAEL
Remarks:
Parental generation
Effect level:
>= 70 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Treatment with Salicynalva by dietary administration in male and female Wistar Han rats at dose levels of circa 15, 30 and 70 mg/kg bw (200, 400 and 1.000 ppm nominal, respectively) revealed no parental toxicity for treatment up to 70 mg/kg bw.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, treatment with Salicynalva by dietary administration in male and female Wistar Han rats at dose levels of circa 15, 30 and 70 mg/kg bw (200, 400 and 1.000 ppm nominal, respectively) revealed no parental, reproduction and developmental toxicity for treatment up to 70 mg/kg bw.

A parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1.000 ppm was established.

The actual test substance intake (based on body weight and food consumption) at 1.000 ppm was 70 to 73 mg substance/kg body weight/day for the males and 79 to 124 mg substance/kg body weight/day for the females.
Executive summary:

In a dietary reproscreen study according to OECD TG 421 several systemic parameters were scored in addition to what is required in the reproscreen in view of the systemic effects seen in the 28-day oral gavage study. The doses were 13 and 16, 28 and 32 and 70 -85 mg/kg bw for males and females, respectively (using the actual intake via food being 134, 284 and 770 ppm (200, 400 and 1000 nominal ppm Salicynalva in feed, respectively).

Body weight and clinical signs were similar to control values. For haematology parameters no treatment related findings were seen up to the highest dose. Macroscopic and microscopic observations did not show treatment related effects. Relative liver weights were increased at 70-80 mg/kg bw in both sexes, circa 10% for males and 20% for females. No organ weight effect were seen in: kidney, spleen, testes and epidymides.Considering systemic effects the NOAEL is considered to be 70 mg/kg bw for both males and females, using the lower value of the males on the intake of the substance. Though the liver weights at this dose are increased in the absence of any and macroscopic and microscopic findings this increase in liver weight is considered to be a non-adverse adaptive change.